Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
  • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
  • C07K2319/23—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a GST-tag
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
  • C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag

Definitions

• the invention is directed to molecules that are members of a polypeptide family referred to as Pellino (also called conserveed Inflammatory Signal Target (CIST)). More particularly, the present invention includes Pellino polypeptides and fragments thereof, the nucleic acids encoding such polypeptides, and fragments thereof, processes for production of recombinant forms of such polypeptides, antibodies generated against these polypeptides, transgenic and knockout cells and animals, and uses thereof.
• Pellino also called conserveed Inflammatory Signal Target (CIST)
• CIST conserveed Inflammatory Signal Target
• the Interleutun-1 (IL-1) pathway is a cellular signaling pathway is that plays a crucial role in the mammalian inflammatory response.
• IL-1 alpha IL-1 beta and IL-1 receptor antagonist (IL-1ra)
• IL-1RI IL-1 receptor Type 1
• IL-1RII IL-1 receptor Type II
• IL-1RI receptor-associated kinase
• Pelle receptor-associated kinase
• Another Drosophila protein, Pellino has been reported to interact with Pelle (Grosshans et al., Mech. Dev. 81:127; 1999).
• Dorsal-ventral polarization in Drosophila embryos depends upon the establishment of a gradient of nuclear localization of the Rel-like transcription factor Dorsal.
• the transcriptional program mediated by Dorsal results from a signaling cascade triggered by binding of an extracellular ligand Spaetzle to its receptor Toll. Intermediates of this signaling cascade include the adaptor protein Tube, the serine/threonine kinase Pelle, and Cactus, a cytosolic binding partner of Dorsal. Signals transmitted by Toll result in the degradation of Cactus, and thereby permit the nuclear importation of Dorsal.
• TIR Toll/IL-1R
• TLRs mammalian toll-like receptors
• Downstream components of the Toll signaling pathway have also been evolutionarily conserved in mammalian TLR and interleukin-1 receptor signaling pathways which culminate in nuclear translocation of the transcription factor Nuclear Factor kappa B (NF-kB).
• Protein kinases IRAK-1 and IRAK2. close homologues of Pelle, are recruited to the activated IL-1R or TLR receptor complexes through the adaptor protein MyD88 and undergo autophosphorylation reactions.
• MyD88 is not a strict analog of Tube, both proteins contain a so-called death domain, and Tube likely serves to mediate signal transmission between Toll and Pelle, to which it binds.
• IRAK subsequently interacts with another adaptor molecule TRAP-6, which is homologous to the recently described D-TRAF.
• Signals downstream of TRAP appear to be divergent, and not all of them are fully understood, but one consequence, in mammalian cells, is the activation of the IkB kinase (IKK) complex which directly phosphorylates the inhibitory Cactus homolog IkB at two N-terminal serine residues causing its ubiquitination and degradation. Released from a cytoplasmic association with IkB, NF-kB migrates into the nucleus. Recently, a candidate for an additional intermediate in Tube-Pelle interactions was found by yeast two-hybrid screening with Pelle as a bait sequence.
• IKK IkB kinase
• Pellino This protein, called Pellino, was shown to interact with catalytically-competent Pelle, but not with a mutant form of Pelle that lacked kinase activity. Although a function for Pellino was not addressed in this study, it was suggested that it could either stabilize the activated form of Pelle, or mediate an interaction with downstream Pelle substrates.
• Pellino-2 A second homologous gene, Pellino-2, was found in humans and mice (Resch et al., Cytogenetics and Cell Genetics (2001) 92(1-2):172-174). In the same publication, a note was added in proof, pointing to indications of the existence of a third human Pellino gene (termed Pellino-3). Kennedy et al. (FASEB J. (2001) 15(7):A209) reported that Pellino-1 (referred to as PRISM) stimulates NF- ⁇ B activation and that a dominant negative Pellino-1 mutant blocks Toll and IL-1 stimulation of NF- ⁇ B.
• PRISM Pellino-1
• IL-1 and other pro-inflammatory cytokines have been implicated in a variety of diseases and conditions, including rheumatoid arthritis, multiple myeloma, osteoporosis, endotoxemia and sepsis, osteoarthritis, inflammatory bowel disease, and allergy. Inhibition of the signaling of IL-1 using soluble forms of IL-1Rs, and the IL-1ra, have been shown to be useful in treating or ameliorating disease characterized by excess levels of IL-1 (Rosenwasser, J. Allergy Clin. Immunol. 102:344; 1998).
• the present invention is based upon the discovery of new murine and human Pellino polypeptides, murine Pellino-1 and -2, and human Pellino-1, -2, and -3.
• the invention provides an isolated polypeptide capable of inhibiting NF-kB-dependent or p38-dependent transcription, the polypeptide comprising an amino acid sequence selected from the group consisting of:
• the invention also provides an isolated genomic nucleic acid corresponding to the nucleic acids of the invention.
• nucleic acids encoding polypeptides of the invention, allelic variants of these nucleic acids
• expression vectors and recombinant host cells comprising at least one nucleic acid of the invention, and preferred recombinant host cells wherein said nucleic acid is integrated into the host cell genome.
• a preferred process provided by the invention further comprises purifying said polypeptide.
• the polypeptide produced by said process is provided.
• in vitro method for inhibiting NF-kB-dependent transcription comprising providing at least one polypeptide of the invention.
• an in vitro method for inhibiting IL-1-mediated p38 kinase signaling comprising providing at least one antagonist of a polypeptide of the invention, wherein the antagonist is an antibody that inhibits the activity of said polypeptide.
• Another aspect of the invention is the use of the polypeptides of the invention as a medicament.
• a further embodiment of the invention provides a use for the "dominant-negative" Pellino polypeptides of the invention in the preparation of a medicament for treating an inflammatory condition; with a preferred embodiment wherein the inflammatory condition is selected from the group consisting of asthma, rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, atherosclerosis, and Alzheimer's disease.
• fusion proteins comprising any of the aforementioned polypeptides and a polypeptide selected from the group consisting of an immunoglobulin Fc domain, a FLAG peptide, a peptide comprising at least about 6 His residues, a leucine zipper, a GFP peptide, a PkA peptide, a birA peptide, and a GST peptide.
• Nucleic acid molecules that encode such fusion proteins are also included within the instant invention, as are recombinant expression vectors comprising any of the aforementioned DNAs, host cells transformed or transfected with such expression vectors, and processes for preparing polypeptides, comprising culturing such host cells under conditions promoting expression, and recovering the polypeptides.
• the invention further provides transgenic or knockout animals generated by using the inventive DNAs..
• the invention further provides antibodies that specifically binds the inventive polypeptides, including monoclonal antibodies and human antibodies. Assays for identification of small molecules that regulate IL-1 signaling, utilizing an inventive peptide, are also provided.
• Pellino polypeptides such as Pellino-1 and Pellino-2 strongly activates NF-kB-dependent reporter genes and augments Jun N-terminal kinase, p38 kinase, and ERK signaling mediated by IL-1, and that mutant forms of Pellino, lacking conserved motifs, suppress basal and cytokine-induced NF-kB activation, and also p38-dependent transcription.
• the molecules of this invention have utility as, or lead to, anti-inflammatory therapies.
• the discovery of the polynucleotides of the invention enables the construction of expression vectors comprising DNA that encodes polypeptides; host cells transfected or transformed with the expression vectors; development of transgenic and knockout cells and animals; isolated and purified polypeptides and fragments thereof; the use of the polynucleotides thereof as probes or primers to identify DNA encoding proteins having Pellino activity, the use of single-stranded sense or antisense oligonucleotides from the nucleic acids to inhibit expression and/or function of polynucleotide encoded by the Pellino genes; the use of such polynucleotides or polypeptides to identify small molecule inhibitors of protein association or function of Pellino; the use of such polynucleotides or polypeptides to identify other molecules involved in IL-1 signaling; the use of such polypeptides and fragments thereof to generate antibodies; and the use of such antibodies to purify the Pellino polypeptide.
• the amino acid sequences of murine and human Pellino-1, murine and human Pellino-2, and human Pellino-3 polypeptide are provided in SEQ ID NOs 2, 4, 6, 8, and 12, respectively, and an alignment showing the sequence similarities between murine and human Pellino-1 and -2, human Pellino-3, and other Pellino polypeptides is presented in Table 1in Example 1 below.
• the Pellino polypeptide family is remarkably well conserved, with the human family members highly similar to each other, and extremely similar to homologous Pellino family members from other species such as Mus musculus.
• Typical structural elements common to members of the Pellino polypeptide family include a particularly well-conserved central domain, extending from amino acid 132 through amino acid 193 in Pellino-1 (SEQ ID NOs 2 and 4; which corresponds to amino acids 134 through 195 in SEQ ID NO:8 and amino acids 158 through 219 in SEQ ID NO:12); an absolutely conserved motif from residue 245 through residue 254 of SEQ ID NOs 2 and 4 (which corresponds to amino acids 247 through 256 in SEQ ID NO:8 and amino acids 271 through 280 in SEQ ID NO:12); and a domain ("the RING-finger-like domain"), similar to the C3HC4 RING-finger subfamily of Zinc-finger domains, from amino acid 333 through amino acid 398 of SEQ ID NOs 2 and 4 (which corresponds to amino acids 335 through 400 in SEQ ID NO:8 and amino acids 360 through 425 in SEQ ID NO:12).
• SEQ ID NOs 2 and 4 which corresponds to
• the conserved cysteine residues within the Pellino polypeptides are located at positions 333, 336, 367, 371, 395, and 398 of SEQ ID NOs 2 and 4 (and at positions 335, 338, 369, 373, 397, and 400 of SEQ ID N0:8 and the corresponding positions in SEQ ID NO:6, and at positions 360, 363, 394, 398, 422, and 425 of SEQ ID NO:12).
• Biological activities or functions associated with murine and human Pellino-1 and -2, and human Pellino-3 polypeptides include stimulation of MAP kinase-activated signaling pathways, such as pro-inflammatory signaling pathways, and in particular stimulation of transcription from downstream promoters such as NF-kB- and p38-dependent promoters.
• the ability of murine and human Pellino-1 and -2 and human Pellino-3 polypeptides to stimulate MAP kinase-activated signaling pathways is associated with many domains of the Pellino polypeptides (such as the N-terminal, central conserved domain, the RING-finger-like domain, and the C-terminal domain) or with the polypeptides in their entirety, as deletions of Nor C-terminal domains and certain modifications of key residues within murine Pellino-1 have been shown either to abolish this stimulatory activity, or to generate "dominant negative" Pellino-1 mutants which inhibit MAP kinase-activated signaling pathways.
• many domains of the Pellino polypeptides such as the N-terminal, central conserved domain, the RING-finger-like domain, and the C-terminal domain
• deletions of Nor C-terminal domains and certain modifications of key residues within murine Pellino-1 have been shown either to abolish this stimulatory activity, or to
• the ability of murine and human Pellino-1 and -2 and human Pellino-3 polypeptides to stimulate MAP kinase-activated signaling pathways can be determined, for example, in an assay that measures the transcription of reporter genes, such as the luciferase coding sequence or the chloramphenicol acetyl transferase (CAT) coding sequence, from downstream promoters, cush as the NF-kB-dependent IL-8 promoter or the p38-dependent CHOP promoter.
• reporter genes such as the luciferase coding sequence or the chloramphenicol acetyl transferase (CAT) coding sequence
• Pellino polypeptides that stimulate MAP kinase-activated signaling pathways preferably have at least 10% (more preferably, at least 25%, and most preferably, at least 50%) of this stimulatory activity as compared to that of murine Pellino-1 measured in the NF-kB-dependent IL-8 promoter-luciferase reporter gene assays of Example 2.
• Murine and human Pellino-1 and -2, and human Pellino-3 polypeptides are also substrates for proteases, such as chymotrypsin-like serine proteases, and demonstrate a change in solubility in response to stimulation of cells by stimulatory molecules such as TNF-alpha and PMA.
• proteases such as chymotrypsin-like serine proteases
• the protease-substrate activity is associated with the central domain of murine and human Pellino-1 and -2 and human Pellino-3 polypeptides, this central domain comprising residues 154 and 165 of SEQ ID NO:2 (or the corresponding residues of other Pellino polypeptides), substitutions to which have been shown to reduce the cleavage of Pellino- 1.
• preferred murine and human Pellino-1 and -2 and human Pellino-3 polypeptides include those comprising residues 154 and 165 of SEQ ID NO:2 (or the corresponding residues of other Pellino polypeptides) or having the conserved central domain, and exhibiting proteolytic cleavage in response to appropriate cell stimuli, such as treatment with TNF-alpha or PMA.
• Preferred murine and human Pellino-1 and -2 and human Pellino-3 polypeptides further include oligomers or fusion polypeptides comprising at least one conserved central domain of one or more murine and human Pellino-1 and -2 and human Pellino-3 polypeptides, and fragments of any of these polypeptides, exhibiting proteolytic cleavage in response to appropriate cell stimuli, such as treatment with TNF-alpha or PMA.
• the protease-substrate activity of murine and human Pellino-1 and -2 and human Pellino-3 polypeptides can be determined, for example, in an assay that measures the extent of Pellino polypeptide cleavage as described in Examples 3 and 4 below.
• Pellino polypeptides having protease-substrate activity preferably have at least 10% (more preferably, at least 25%, and most preferably, at least 50%) of the protease-substrate activity of murine Pellino-1-FLAG as measured in the assays of Examples 3 and 4.
• Biological activities or functions associated with certain mutant or altered forms of murine and human Pellino-1 and -2, and human Pellino-3 polypeptides include inhibition of MAP kinase-activated signaling pathways, such as pro-inflammatory signaling pathways, and in particular inhibition of transcription from downstream promoters such as NF-kB- and p38-dependent promoters.
• the ability of altered murine and human Pellino-1 and -2 and human Pellino-3 polypeptides to inhibit MAP kinase-activated signaling pathways can be determined, for example, in an assay that measures the transcription of reporter genes, such as the luciferase coding sequence or the chloramphenicol acetyltransferase (CAT) coding sequence, from downstream promoters, such as the NF-kB-dependent IL-8 promoter or the p38-dependent CHOP promoter.
• reporter genes such as the luciferase coding sequence or the chloramphenicol acetyltransferase (CAT) coding sequence
• CAT chloramphenicol acetyltransferase
• Pellino polypeptides that inhibit MAP kinase-activated signaling pathways preferably have at least 10% (more preferably, at least 25%, and most preferably, at least 50%) of this inhibitory activity as compared to that of the murine Pellino-1-FLAG "d133-156-FLAG" mutant as measured in the NF-kB-dependent IL-8 promoter-luciferase reporter gene assays of Example 2.
• Pellino polypeptide activity includes any one or more of the following: stimulation of MAP kinase-activated signaling pathways, protease-substrate activity, and host defensive activity against pathogens, as well as the ex vivo and in vivo activities of wild type Pellino polypeptides.
• Pellino polypeptide dominant-negative activity includes inhibition of MAP kinase-activated signaling pathways, anti-inflammatory activity, and the ability to sequester binding partners in the insoluble cell fraction, as well as the ex vivo and in vivo activities of mutant Pellino polypeptides that demonstrate such inhibitory activities in reporter gene assays.
• the degree to which individual members of the Pellino polypeptide family and fragments and other derivatives of these polypeptides exhibit these activities can be determined by standard assay methods, particularly assays such as those described in Examples 2, 3. and 4 below. Exemplary assays are disclosed herein; those of skill in the art will appreciate that other, similar types of assays can be used to measure Pellino polypeptide biological activities.
• Pellino polypeptides Another aspect of the biological activity of Pellino polypeptides is their ability to interact with particular intracellular signaling pathway molecules such as TRAF2, TRAF6, IRAK, TRAF1, and TRAFs 3, 4, and 5, with the RING-finger-like domain of Pellino polypeptides likely involved in binding to such binding partners.
• the conserved central domain of Pellino polypeptides interacts with a chymotrypsin-like serine protease that cleaves Pellino polypeptides, and the N-terminal portion of Pellino polypeptides is believed to bind a factor involved in localizing Pellino peptides in, or transporting them to, the portion of the cellular environment that becomes the soluble fraction upon cell lysis.
• binding partner includes ligands, receptors, substrates, antibodies, other Pellino polypeptides, the same Pellino polypeptide (in the case of homotypic interactions), and any other molecule that interacts with a Pellino polypeptide through contact or proximity between particular portions of the binding partner and the Pellino polypeptide.
• the RING-finger-like domain of Pellino polypeptides is believed to bind to a signaling pathway binding partner, the RING-finger-like domain when expressed as a separate fragment from the rest of a Pellino polypeptide, but with enough of the N-terminal domain to allow the Pellino polypeptide to localize to the insoluble fraction, is expected to disrupt the binding of wild-type Pellino polypeptides to their binding partners.
• BRET bioluminescence resonance energy transfer
• the partner-binding activities of Pellino polypeptides can be determined using protein-fragment complementation assays, as described in Remy and Michnick, 1999, Proc Natl Acad Sci USA 96: 5394-5399 and in WO 01/00866.
• Pellino polypeptides such as murine and human Pellino-1 and -2 and human Pellino-3 polypeptides with the ability to stimulate MAP kinase-activated pathways are believed to play a role in protection of the host against viral, bacterial, fungal, and other types of pathogens (innate immune responses).
• Pellino polypeptides are involved in immune and/or inflammatory diseases or conditions, that share as a common feature stimulation of MAP kinase-activated pathways and NF-kB- and/or p38-dependent transcription in their etiology.
• the therapeutic effect of stimulation of MAP kinase-activated pathways for example by administration of a Pellino polypeptide with wild-type activity, or fragments or fusion polypeptides with wild-type activity, or agonists thereof, is shown by the following examples of conditions in which the stimulation of NF-kB-dependent transcription is beneficial (Yamamoto and Gaynor, 2001, J Clin Invest 107: 135-142).
• the NF-kB pathway modulates B-lymphocyte survival, mitogen-dependent cell proliferation, and isotype switching, which lead to the differentiation of B lymphocytes into plasma cells.
• NF- ⁇ B regulates IL-2 production, which increases the proliferation and differentiation of T lymphocytes, and increases the development of Th1-type helper T cells, promoting cell-mediated immunity.
• activation of NF-kB leads to the induction of multiple genes that regulate the immune response.
• the NF-kB pathway is also a key mediator of genes involved in the control of the cellular proliferation and apoptosis.
• Antiapoptotic genes that are directly activated by NF-kB include the cellular inhibitors of apoptosis (c-IAP1, c-IAP2, and IXAP), the TNF receptor-associated factors (TRAF1 and TRAF2), the Bcl-2 homologue A1/Bf1-1, and IEX-IL.
• antiapoptotic proteins block the activation of caspase-8, an initiator protease, involved at an early step in stimulating the apoptotic pathway, and induction of A1/Bf1-1 expression by NF-kB prevents cytochrome c release from mitochondria and activation of caspase-3.
• NF-kB activation can thus reduce apoptosis in response to treatment with different chemotherapeutic agents.
• NF-kB is involved in protecting cells from undergoing apoptosis in response to DNA damage or cytokine treatment.
• NF-kB regulates host inflammatory responses by increasing the expression of specific cellular genes, including genes encoding at least 27 different cytokines and chemokines.
• Cytokines that are stimulated by NF-kB can also directly activate the NF-kB pathway, thus establishing a positive autoregulatory loop that can amplify the inflammatory response and increase the duration of chronic inflammation.
• NF-kB also stimulates the expression of enzymes whose products contribute to the pathogenesis of the inflammatory process, including the inducible form of nitric oxide synthase (iNOS), which generates nitric oxide (NO), and the inducible cyclooxygenase (COX-2), which generates prostanoids.
• iNOS nitric oxide synthase
• COX-2 inducible cyclooxygenase
• NF-kB pathway Activation of the NF-kB pathway is involved in the pathogenesis of chronic inflammatory diseases, such as asthma, rheumatoid arthritis, and inflammatory bowel disease, and other diseases in which inflammation plays a role, such as atherosclerosis and Alzheimer's disease.
• chronic inflammatory diseases such as asthma, rheumatoid arthritis, and inflammatory bowel disease
• NF-kB activation of cytokine genes is an important contributor to the pathogenesis of asthma, which is characterized by the infiltration of inflammatory cells and the dysregulation of many cytokines and chemokines in the lung.
• Cytokines such as TNF-alpha, that activate NF-kB are elevated in the synovial fluid of patients with rheumatoid arthritis and contribute to the chronic inflammatory changes and synovial hyperplasia seen in the joints of these patients. Increases in the production of proinflammatory cytokines by both lymphocytes and macrophages has also been implicated in the pathogenesis of inflammatory bowel diseases, including Crohn's disease and ulcerative colitis. NF-kB activation is seen in mucosal biopsy specimens from patients with active Crohn's disease and ulcerative colitis. Treatment of patients with inflammatory bowel diseases with steroids decreases NF-kB activity in biopsy specimens and reduces clinical symptoms.
• Atherosclerosis is triggered by numerous insults to the endothelium and smooth muscle of the damaged vessel wall.
• a large number of growth factors, cytokines, and chemokines released from endothelial cells, smooth muscle, macrophages, and lymphocytes are involved in this chronic inflammatory and fibroproliferative process.
• Regulation of genes involved in the inflammatory response and in the control of cellular proliferation by NF-kB likely plays an important role in the initiation and progression of atherosclerosis.
• Abnormalities in the regulation of the NF-kB pathway may be involved in the pathogenesis of Alzheimer's disease.
• NF-kB immunoreactivity is found predominantly in and around early neuritic plaque types in Alzheimer's disease, whereas mature plaque types show vastly reduced NF-kB activity.
• NF-kB activation may be involved in the initiation of neuritic plaques and neuronal apoptosis during the early phases of Alzheimer's disease.
• Other conditions in which inflammation plays a role and which are expected to be ameliorated by decreases in MAP kinase-activated pro-inflammatory signaling pathways include osteoporosis, stroke, multiple sclerosis, and multiple myeloma. Additional examples of diseases involving inflammation and/or inflammatory cellular responses are described U.S. Patent No. 6,204,261 at column 206, line 25, through column 207, line 44.
• constitutive activation of the NF-kB pathway has also been implicated in the pathogenesis of some human cancers.
• Abnormalities in the regulation of the NF-kB pathway are frequently seen in a variety of human malignancies including leukemias, lymphomas, and solid tumors. These abnormalities result in constitutively high levels of NF-kB in the nucleus of a variety of tumors including breast, ovarian, prostate, and colon cancers. The majority of these changes are likely due to alterations in regulatory proteins that activate signaling pathways that lead to activation of the NF-kB pathway.
• Preventing, blocking, and/or inhibiting the interactions between Pellino polypeptides and their binding partners is an aspect of the invention and provides methods for treating or ameliorating these diseases and conditions through the use of inhibitors of wild-type Pellino activities such as stimulation of NF- ⁇ B-dependent transcription.
• the invention in a particular embodiment, relates to certain isolated polynucleotide molecules that are free from contaminating endogenous material.
• Polynucleotide molecule refers to polynucleotide molecules in the form of separate fragments or as a component of larger polynucleotide constructs.
• the polynucleotide molecules have preferably been derived from DNA or RNA isolated at least once in substantially pure form and in a quantity or concentration enabling identification, manipulation, and recovery of its component nucleotide sequences by standard biochemical methods (such as those outlined in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed.. Cold Spring Harbor Laboratory, Cold Spring Harbor. NY (1989)).
• sequences are preferably provided and/or constructed in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, that are typically present in eukaryotic genes. Sequences of non-transtated DNA can be present 5' or 3' from an open reading frame, where the same do not interfere with manipulation or expression of the coding region.
• Polynucleotide molecules of the invention include DNA in both single-stranded and doublestranded form, as well as the corresponding complementary sequences.
• DNA includes, for example, cDNA. genomic DNA , chemically synthesized DNA, DNA amplified by PCR, and combinations thereof. Genomic DNA may be isolated by conventional techniques, e.g., using the cDNA of SEQ ID NOs:1, 3, 5, or 7, or a suitable fragment thereof, as a probe.
• the DNA molecules of the invention include DNAs encoding full length Pellino polypeptides as well as polynucleotides and fragments thereof.
• the polynucleotides of the invention are preferentially derived from human sources, but the invention includes those derived from non-human species, as well.
• the present application describes murine Pellino-1 DNA having the polynucleotide sequence of SEQ ID NO:1 and the polypeptide encoded by the DNA of SEQ ID NO:1 having the amino acid sequence of SEQ ID NO:2.
• the polypeptide having amino acids 132 through 189 of SEQ ID NO:2 is a target site for protease action for a member of the chymotrypsin family of proteases.
• the present application further describes human Pettino-1 DNA having the polynucleotide sequence of SEQ ID N0:3 and the polypeptide encoded by the DNA of SEQ ID NO:3 having the amino acid sequence of SEQ ID NO:4.
• a specific protease target site is alsolound this polypeptide, corresponding to amino acids 132 through 189 of SEQ ID NO:4. Furtherdescribed is the DNA of murine Pellino-2 and having the polynucleotide sequence of SEQ ID NO:5. and polypeptide encoded by SEQ ID NO:5 shown in SEQ ID NO:6.
• the protease target site is located between amino acids 133 and 190 of murine Pellino-2.
• the protease target site of human Pellino-2 is likely to be between amino acids 134 and 191 of SEQ ID NO:8.
• SEQ ID NO:4, and SEQ ID N0:6 may vary by one or more amino acids.
• amino terminus of the protease target region for SEQ ID NO:2 and SEQ ID NO:4 may occur from amino acid 130 through 134 and the carboxy terminus of the target region from amino acid 187 through 191.
• amino terminus of the protease target region occurs from amino acid 131 through amino acid 135 (amino acids 132 through 136 of SEQ ID NO:8) and the carboxy terminus from amino acid 188 through 192 (amino acids 189 through 193 of SEQ ID NO:8).
• a DNA can vary from that shown in SEQ ID NO:1, and still encode a polypeptide having the amino acid sequence of SEQ ID NO:2.
• Such variant DNAs can result from silent mutations that occur naturally, or during PCR amplification, or they can be the product of deliberate mutagenesis of a native sequence. The same is true for the DNAs depicted in SEQ ID NOs: 3, 5 and 7.
• the application thus describes isolated DNAs encoding polypeptides of the invention, selected from: (a) a DNA comprising the nucleotide sequence of SEQ ID NO:1: (b) a DNA comprising the nucleotide sequence of SEQ ID NO:3; (c) a DNA comprising the nucleotide sequence of SEQ ID NO.5; (d) a DNA encoding the polypeptides encoded by the DNA of (a), (b) or (c); (e) a DNA capable of hybridization to the DNA of (a).
• one way of achieving moderately stringent conditions involves the use of a prewashing solution containing 5XSSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization buffer of about 50% formamide, 6XSSC, and a hybridization temperature of about 42°C (or other similar hybridization solutions, such as one containing about 50% formamide, with a hybridization temperature of about 42°C), and washing conditions of about 60°C, in 0.5XSSC, 0.1% SDS.
• Conditions of high stringency can also be readily determined by the skilled artisan based on, for example, the length and base composition of the DNA.
• hybridization conditions are defined as hybridization conditions as above, but with washing at approximately 68°C. 0.2XSSC, 0.1% SDS. It should be understood that the wash temperature and wash salt concentration can be adjusted as necessary to achieve a desired degree of stringency by applying the basic principles that govern hybridization reactions and duplex stability, as known to those skilled in the art (see, e.g., Sambrook et al., 1989). It should be further understood that hybridization conditions for oligonucleotide probes of defined length and sequence can be designed by applying formulae known in the art (e.g., see Sambrook et al., 1989, at 11.4511.47).
• DNA encoding polypeptide fragments that have at least one activity of Pellino polypeptides, and DNA encoding polypeptides of at least about 16 amino acids, or of at least about 32 amino acids, which polypeptides are useful as immunogens.
• DNAs encoding polypeptides comprising inactivated N-glycosylation site(s), inactivated protease processing site(s), or conservative amino acid substitution(s), are also included, as described below.
• the IL,IR-homologous domain may be useful as a dominant negative regulator of IL-1R signaling, or in an assay to identify small molecules that can inhibit or otherwise regulate IL-1 signaling.
• the DNA molecules described in the application also comprises polynucleotides that are at least 80% identical to a native sequence, and polynucleotide molecules that are at least 85% identical to a native molecule. Also contemplated are embodiments in which a DNA molecule is at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or at least 99.9% identical to a native sequence. Percent identity is defined as the number of aligned symbols, i.e. nucleotides or amino acids, which are identical, divided by the total number of symbols in the shorter of the two sequences. The degree of homology (percent identity) between two sequences may be determined by using the alignment method of Needleman and Wunsch ( J . MoL Diol.
• the comparison is done using a computer program.
• An exemplary, preferred computer program is the Genetics Computer Group (GCG; Madison, WI) Wisconsin package version 10.0 program.
• the preferred default parameters for the 'GAP' program includes: (1) The GCG implementation of the previously stated comparison matrixes for nucleotides and amino acids; (2) a penalty of 30 for each gap and an additional penalty of 1 for each symbol in each gap for amino acid sequences, or penalty of 50 for each gap and an additional penalty of 3 for each symbol in each gap for nucleotide sequences; (3) no penalty for end gaps; and (4) no maximum penalty for long gaps.
• Other programs used by one skilled in the art of sequence comparison may also be used.
• DNAs of the invention include variants that differ from a native DNA sequence because of one or more deletions, insertions or substitutions, but that encode a biologically active polypeptide.
• DNAs that encode various additions or substitutions of amino acid residues or sequences, or deletions of terminal or internal residues or sequences are encompassed by the invention.
• DNAs examples include those that have been modified to facilitate expression of a polypeptide with an altered N-linked glycosylation site or KEX-2 protease site, as well as those in which codons that encode Cys residues that are not necessary for biological activity are eliminated or altered to encode another amino acid. These and other variant peptides are disclosed herein; DNAs encoding them are also encompassed by the invention.
• the invention also provides isolated DNAs useful in the production of polypeptides.
• polypeptides may be prepared by any of a number of conventional techniques.
• a DNA sequence encoding a Pellino polypeptide, or desired fragment thereof may be subcloned into an expression vector for production of the polypeptide or fragment.
• the DNA sequence advantageously is fused to a sequence encoding a suitable leader or signal peptide.
• the desired DNA fragment may be chemically synthesized using known techniques.
• DNA fragments also may be produced by restriction endonuclease digestion of a full length cloned DNA sequence, and isolated by electrophoresis on agarose gels.
• oligonucleotides that reconstruct the 5' or 3' terminus to a desired point may be ligated to a DNA fragment generated by restriction enzyme digestion.
• Such oligonucleotides may additionally contain a restriction endonuclease cleavage site upstream of the desired coding sequence, and position an initiation codon (ATG) at the N-terminus of the coding sequence.
• ATG initiation codon
• PCR polymerase chain reaction
• Oligonucleotides that define the desired termini of the DNA fragment are employed as 5' and 3' primers.
• the oligonucleotides may additionally contain recognition sites for restriction endonucleases, to facilitate insertion of the amplified DNA fragment into an expression vector.
• PCR techniques are described in Saiki et al., Science 239:487 (1988); Recombinant DNA Methodology, Wu et al., eds., Academic Press, Inc., San Diego (1989), pp. 189-196; and PCR Protocols: A Guide to Methods and Applications, Innis et al., eds., Academic Press, Inc. (1990).
• the present invention also provides genes corresponding to the nucleic acid sequences disclosed herein.
• “Corresponding genes” or “corresponding genomic nucleic acids” are the regions of the genome that are transcribed to produce the mRNAs from which cDNA nucleic acid sequences are derived and can include contiguous regions of the genome necessary for the regulated expression of such genes. Corresponding genes can therefore include but are not limited to coding sequences, 5' and 3' untranslated regions, alternatively spliced exons, introns, promoters, enhancers, and silencer or suppressor elements.
• Corresponding genomic nucleic acids can include 10000 basepairs (more preferably, 5000 basepairs, still more preferably, 2500 basepairs, and most preferably, 1000 basepairs) of genomic nucleic acid sequence upstream of the first nucleotide of the genomic sequence corresponding to the initiation codon of the Pellino polypeptide coding sequence, and 10000 basepairs (more preferably, 5000 basepairs, still more preferably, 2500 basepairs, and most preferably, 1000 basepairs) of genomic nucleic acid sequence downstream of the last nucleotide of the genomic sequence corresponding to the termination codon of the Pellino polypeptide coding sequence.
• the corresponding genes or genomic nucleic acids can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic materials.
• An "isolated gene” or “an isolated genomic nucleic acid” is a genomic nucleic acid that has been separated from the adjacent genomic sequences present in the genome of the organism from which the genomic nucleic acid was isolated.
• the invention encompasses polypeptides and fragments thereof in various forms, including those that are naturally occurring or produced through various techniques such as procedures involving recombinant DNA technology. Such forms include, but are not limited to, derivatives, variants, and oligomers, as well as fusion proteins or fragments thereof.
• polypeptides described herein include full length proteins encoded by the nucleic acid sequences set forth above.
• Full length polypeptides comprise an amino acid sequence as depicted in SEQ ID NOs: 2, 4, and 6, with useful fragments comprising amino acids 132 to 289 of SEQ ID NOs:2 and 4, and amino acids 133 to 190 of SEQ ID NO:6.
• the N-terminal and C-terminal amino acids of these and other fragments can vary about two amino acids from those given (i.e., the N-terminus can vary from amino acids 130 to 134 of SEQ ID NOs:2 and 4 and 131 to 135 of SEQ ID NO:6; and the C-terminus can vary from amino acids 187 to 191 of SEQ ID NOs:2 and 4 and 188 to 192 of SEQ ID NO:6).
• inventive peptides and fragments thereof may be recombinantly expressed as an intracellular polypeptide, preferably in non-mammalian cells.
• Such peptides may be obtained by isolating cells that express the polypeptide from the culture medium (e.g., by centrifugation or filtration), solubilizing the cells, and isolating the peptide from the solubilized cells. Choice of solubilization techniques will depend on the cells used for expression. Purification of the polypeptide from recombinant host cells is facilitated by expression of the polypeptide as a fusion protein with a tag protein as discussed herein.
• inventive peptides and fragments thereof may also be recombinantly expressed as a soluble polypeptide capable of being secreted from the cells in which it is made.
• soluble peptides may be obtained by separating intact cells that express the soluble polypeptide from the culture medium (e.g., by centrifugation or filtration), and isolating the soluble peptide from the medium (supernatant). Purification of the polypeptides from recombinant host cells is facilitated by expression of the polypeptide as a secreted protein, which can be useful in obtaining large amounts of the soluble polypeptide as a therapeutic or diagnostic agent, or for use in assays. Because the N-terminus and C-terminus of recombinantly expressed polypeptides may vary by several amino acids, including from about 1 amino acid to about 10 amino acids, the polypeptides of this invention can vary accordingly.
• the invention also provides Pellino polypeptides and fragments thereof that retain a desired activity. Particular embodiments are directed to polypeptide fragments that retain the ability to bind a member of the chymotrypsin family of proteases. Such a fragment may be a soluble polypeptide, as described above. In another embodiment, the polypeptides and fragments advantageously include regions that are conserved in the Pellino family as described above.
• polypeptide fragments comprising at least 8, 12. 16. or at least 32, contiguous amino acids of the sequence of SEQ ID NO:2. Such polypeptide fragments may be employed as immunogens in generating antibodies, as small molecule agonists or antagonists of Pellino activity, and in various assays for Pellino activity.
• variants as well as derived variants of the polypeptides and fragments are provided herein.
• Variants may exhibit amino acid sequences that are at least 80% identical, or at least about 85% identical, to the native polypeptide disclosed herein.
• a polypeptide or fragment comprises an amino acid sequence that is at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or at least 99.9% identical to the preferred polypeptide or fragment thereof. Percent identity may be determined as described previously herein.
• variants of the invention include, for example, those that result from alternate mRNA splicing events or from proteolytic cleavage.
• Alternate splicing of mRNA may, for example, yield a truncated but biologically active protein, such as a naturally occurring, shortened form of the protein.
• variations attributable to proteolysis include, for example, differences in the N- or C-termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the protein (generally from about one to about five terminal amino acids) or other differences in protein expression. Proteins in which differences in amino acid sequence are attributable to genetic polymorphism (allelic variation among individuals producing the protein) are also contemplated herein.
• fusion proteins such as those prepared by expression in recombinant culture as N-terminal or C-terminal fusions.
• fusion proteins include fusion proteins that will form oligomers, such as a Pellino/Fc fusion protein (for example, as described in U.S. Patent 5,962,406, issued October 5. 1999), or a zipper fusion protein (U.S. Patent 5,716,805, issued February 10, 1998).
• fusion proteins can comprise peptides added to facilitate purification and identification (often referred to as tag proteins).
• tag proteins include, for example, poly-His or the antigenic identification peptides described in U.S. Patent No.
• GFP green fluorescent protein
• PKA site-specific protein kinase
• GST glutathione S transferase
• FLAG peptide is highly antigenic and provides an epitope reversibly bound by a specific monoclonal antibody, enabling rapid assay and facile purification of expressed recombinant protein.
• a murine hybridoma designated 4E11 produces a monoclonal antibody that binds the FLAG peptide in the presence of certain divalent metal cations, as described in U.S. Patent 5,011,912
• the 4E11 hybridoma cell line has been deposited with the American Type Culture Collection under accession no. HB 9259.
• Monoclonal antibodies that bind the FLAG peptide are available from Eastman Kodak Co., Scientific Imaging Systems Division, New Haven, Connecticut.
• Another useful tag peptide is the GST peptide, which binds glutathione, also facilitating purification of expressed recombinant protein.
• Recombinant protein can be purified by affinity chromatography using a suitable chromatography matrix to which has been attached glutathione, as described in Smith and Johnson, supra Suitable chromatography matrixes include Glutathione-Agarose beads (Pharmacia). Recombinant protein can be eluted with an excess of glutathione.
• a specific enzymatic cleavage site (such as a thrombin cleavage site) can be included in the recombinant fusion protein, and the desired polypeptide removed from the affinity matrix by treatment with the enzyme that cleaves the fusion protein at the cleavage site.
• variant polypeptides are variants of native polypeptides that retain the native biological activity or the substantial equivalent thereof.
• One example is a variant that binds a binding partner with essentially the same binding affinity as does the native form. Binding affinity can be measured by conventional procedures, e.g., as described in U.S. Patent No. 5,512,457 and as set forth below.
• Variants include polypeptides that are substantially homologous to the native form, but which have an amino acid sequence different from that of the native form because of one or more deletions, insertions or substitutions, Particular embodiments include, but are not limited to, polypeptides that comprise from one to ten deletions, insertions or substitutions of amino acid residues, when compared to a native sequence.
• a given amino acid may be replaced, for example, by a residue having similar physiochemical characteristics.
• physiochemically conservative substitutions include substitution of one aliphatic residue for another, such as Ile, Val, Leu, or Ala for one another; substitutions of one polar residue for another, such as between Lys and Arg, Glu and Asp, or Gln and Asn; or substitutions of one aromatic residue for another, such as Phe, Trp, or Tyr for one another.
• Other substitutions e.g., involving substitutions of entire regions having similar hydrophobicity characteristics, are well known.
• the invention further induces polypeptides of the invention with or without associated native-pattern glycosylation.
• Polypeptides expressed in yeast or mammalian expression systems e.g., CHO or COS-7 cells
• yeast or mammalian expression systems e.g., CHO or COS-7 cells
• a given preparation may include multiple differentially glycosylated species of the protein.
• Expression of polypeptides of the invention in bacterial expression systems, such as E. coli. provides non-glycosylated molecules. Glycosyl groups can also be removed through conventional chemical or enzymatic methods, in particular those utilizing glycopeptidase.
• glycosylated polypeptides of the invention can be incubated with a molar excess of glycopeptidase (Bochringer Mannheim).
• Recombinant technology can also be applied to reduce glycosylation that occurs in eukaryotic expression systems, for example, as described in U.S. Patent 5,071,972 and EP 276,846.
• Other variants are prepared by modification of adjacent dibasic amino acid residues, to enhance expression in yeast systems in which KEX2 protease activity is present, as disclosed in EP 212,914.
• sequences encoding Cys residues that are not essential for biological activity can be altered to cause the Cys residues to be deleted or replaced with other amino acids, as disclosed in U.S. Patent 5,962,406, issued October 5, 1999.
• polypeptides that may be modified to create derivatives thereof by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like
• Covalent derivatives may be prepared by linking the chemical moieties to functional groups on amino acid side chains or at the N-terminus or C-terminus of a polypeptide.
• Conjugates comprising diagnostic (detectable) or therapeutic agents attached thereto are contemplated herein, as discussed in more detail below.
• the present invention also provides recombinant cloning and expression vectors containing DNA, as well as host cell containing the recombinant vectors.
• Expression vectors comprising DNA may be used to prepare the polypeptides or fragments of the invention encoded by the DNA.
• a method for producing polypeptides comprises culturing host cells transformed with a recombinant expression vector encoding the polypeptide, under conditions that promote expression of the polypeptide, then recovering the expressed polypeptides from the culture.
• the skilled artisan will recognize that procedures for producing and purifying the expressed polypeptides will vary according to such factors as the type of host cells employed, and whether the polypeptide is membrane-bound or a soluble polypeptide that is secreted from the host cell.
• the vectors include a DNA encoding a polypeptide or fragment of the invention, operably linked to suitable transcriptional or translational regulatory nucleotide sequences, such as those derived from a mammalian, microbial, viral, or insect gene.
• suitable transcriptional or translational regulatory nucleotide sequences such as those derived from a mammalian, microbial, viral, or insect gene.
• regulatory sequences include uanscriptional promoters, operators, or enhancers, an mRNA ribosomal binding site, and appropriate sequences which control transcription and translation initiation and termination.
• An origin of replication that confers the ability to replicate in the desired host cells, and a selection gene by which transformants are identified, are generally incorporated into the expression vector.
• Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the DNA sequence.
• a promoter nucleotide sequence is operably linked to a DNA sequence if the promoter nucleotide sequence controls the transcription of the DNA
• a sequence encoding an appropriate signal peptide can be incorporated into expression vectors.
• a DNA sequence for a signal peptide may be fused in frame to the nucleic acid sequence of the invention so that the DNA is initially transcribed, and the mRNA translated, into a fusion protein comprising the signal peptide.
• a signal peptide that is functional in the intended host cells promotes extracellular secretion of the polypeptide. The signal peptide is cleaved from the polypeptide upon secretion of polypeptide from the cell.
• a protein preparation may include a mixture of protein molecules having different N-terminal amino acids, resulting from cleavage of the signal peptide at more than one site.
• Suitable host cells for expression of polypeptides include prokaryotes, yeast or higher eukaryotic cells.
• Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described, for example, in Pouwels et al. Cloning Vectors: A Laboratory Manual, Elsevier, New York, (1985). Cell-free translation systems could also be employed to produce polypeptides using RNAs derived from DNA constructs disclosed herein.
• Mammalian or insect host cell culture systems also may be employed to express recombinant polypeptides.
• Bacculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47 (1988).
• Established cell lines of mammalian origin also may be employed.
• suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, and BHK (ATCC CRL 10) cell lines, and the CV1/EBNA cell line derived from the African green monkey kidney cell line CV1 (ATCC CCL 70) as described by McMahan et al. (EMBO J. 10: 2821, 1991).
• DHFR dihydrofolate reductase
• DXB11 dihydrofolate reductase
• Another exemplary DHFR- CHO cell line is DG44 (see, for example, Kaufman, R.J., Meth. Enzymology 185:537 (1988).
• Other cell lines developed for specific selection or amplification schemes will also be useful with the invention.
• transfection protocols are known in the art, and are reviewed in Kaufman, R.J., supra.
• the transfection protocol chosen will depend on the host cell type and the nature of the gene of interest, and can be chosen based upon routine experimentation. The basic requirements of any such protocol are first to introduce DNA encoding the protein of interest into a suitable host cell, and then to identify and isolate host cells which have incorporated the heterologous DNA in a stable, expressible manner. Other useful transfection protocols are discussed in U.S. Patent 6,027,915, issued February 22, 2000.
• Transfection of cells with heterologous DNA and selection for cells that have taken up the heterologous DNA and express the selectable marker results in a pool of transfected cells. Individual cells in these pools will vary in the amount of DNA incorporated and in the chromosomal location of the transfected DNA. To generate stable cell lines, individual cells can be isolated from the pools and cultured (a process referred to as cloning).
• a method of amplifying the gene of interest is also desirable for expression of the recombinant protein, and typically involves the use of a selection marker (reviewed in Kaufman, R.J., supra). Resistance to cytotoxic drugs is the characteristic most frequently used as a selection marker, and can be the result of either a dominant trait (i.e., can be used independent of host cell type) or a recessive trait (i.e., useful in particular host cell types that are deficient in whatever activity is being selected for).
• Several amplifiable markers are suitable for use in the inventive expression vectors (for example, as described in Maniatis, Molecular Biology: A Laboratory Manual, Cold Spring Harbor Laboratory, NY, 1989; pgs 16.9-16.14).
• DHFR-MTX resistance P-glycoprotein and multiple drug resistance (MDR)-various lipophilic cytoxic agents (i.e., adriamycin, colchicine, vincristine), and adenosine deaminase (ADA)-Xyl-A or adenosine and 2'-deoxycoformycin.
• MDR multiple drug resistance
• ADA adenosine deaminase
• Other dominant selectable markers are discussed in U.S. Patent 6,027,915, issued February 22, 2000).
• Useful regulatory elements can also be included in the plasmids or expression vectors used to transfect mammalian cells.
• the transfection protocol chosen, and the elements selected for use therein, will depend on the type of host cell used. Those of skill in the art are aware of numerous different protocols and host cells, and can select an appropriate system for expression of a desired protein, based on the requirements of their selected cell culture system(s).
• a useful high expression vector, pCAVNOT has been described by Mosley et al., Cell 59:335-34g, 1989.
• Other expression vectors for use in mammalian host cells can be constructed as disclosed by Okayama and Berg (Mol. Cell. Biol. 3:280. 1983).
• a useful system for stable high level expression of mammalian cDNAs in C127 murine mammary epithelial cells can be constructed substantially as described by Cosman et al. (Mol. Immunol. 23:935, 1986).
• the vectors can be derived from retroviruses.
• pFLAG and pDC311 can also be used.
• FLAG technology is centered on the fusion of a low molecular weight (lkD), hydrophilic.
• pDC311 is another specialized vector used for expressing proteins in CHO cells
• pDC311 is characterized by a bicistronic sequence containing the gene of interest and a dihydrofolate reductase (DHFR) gene with an internal ribosome binding site for DHFR translation, an expression augmenting sequence element (EASE), the human CMV promoter, a tripartite leader sequence, and a polyadenylation site.
• EASE expression augmenting sequence element
• a signal peptide may be employed to facilitate secretion of the protein, if desired.
• the choice of signal peptide or leader may depend on factors such as the type of host cells in which the recombinant polypeptide is to be produced.
• heterologous signal peptides that are functional in mammalian host cells include the signal sequence for interleukin-7 (IL-7) described in United States Patent 4,965,195; the signal sequence for interleulcin-2 receptor described in Cosman et al., Nature 312:768 (1984); the interleukin-4 receptor signal peptide described in EP 367,566; the type I interleukin-1 receptor signal peptide described in U.S. Patent 4,968,607; and the type II interleukin-1 receptor signal peptide described in EP 460,846.
• IL-7 interleukin-7
• the "isolated" polypeptides or fragments thereof encompassed by this invention are polypeptides or fragments that are not in an environment identical to an environment in which it or they can be found in nature.
• the "purified” polypeptides or fragments thereof encompassed by this invention are essentially free of association with other cellular components, such as unrelated proteins or polypeptides, lipids and DNA or RNA, for example, as a purification product of recombinant expression systems such as those described above or as a purified product from a non-recombinant source such as naturally occurring cells and/or tissues.
• the purification of recombinant polypeptides or fragments can be accomplished by expressing the inventive polypeptide(s) as a fusion protein with a peptide (often referred to as a tag peptide) for which an affinity purification scheme is known in the art.
• a peptide often referred to as a tag peptide
• Such fusion partners can include the poly-His or other tag peptides described above as well as an Fc moiety or a zipper moiety.
• procedures for purifying a recombinant polypeptide or fragment will vary according to such factors as the type of host cells employed and whether or not the recombinant polypeptide or fragment is secreted into the culture medium.
• the recombinant polypeptide or fragment can be isolated from the host cells if not secreted, or from the medium or supernatant if soluble and secreted, followed by one or more concentration, salting-out, ion exchange, hydrophobic interaction, affinity purification or size exclusion chromatography steps.
• the culture medium first can be concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
• a purification matrix such as a gel filtration medium.
• an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups.
• the matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification.
• a cation exchange step can be employed.
• Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups.
• a chromatofocusing step can be employed.
• a hydrophobic interaction chromatography step can be employed.
• Suitable matrices can be phenyl or octyl moieties bound to resins.
• affinity chromatography with a matrix which selectively binds the recombinant protein can be employed. Examples of such resins employed are lectin columns, dye columns, and metal-chelating columns.
• RP-HPLC reversed-phase high performance liquid chromatography
• hydrophobic RP-HPLC media e.g., silica gel or polymer resin having pendant methyl, octyl, octyldecyl or other aliphatic groups
• RP-HPLC media e.g., silica gel or polymer resin having pendant methyl, octyl, octyldecyl or other aliphatic groups
• an affinity column comprising a polypeptide-binding protein of the invention, such as a monoclonal antibody generated against polypeptides of the invention, to affinity-purify expressed polypeptides.
• binding proteins such as antibodies against Pellino or other molecules that bind Pellino can be bound to a solid phase support such as a column chromatography matrix or a similar substrate suitable for identifying, separating, or purifying Pellino.
• Adherence of Pellino to a solid phase contacting surface can be accomplished by any means, for example, magnetic microspheres can be coated with Pellino binding proteins (or other Pellino-binding molecules) and held in the incubation vessel through a magnetic field.
• Solutions containing Pellino polypeptides are contacted with the solid phase under conditions promoting binding of Pellino polypeptides to the binding partner; unbound material is then washed away.
• Methods of releasing positively selected peptides from the solid phase are known in the art and encompass, for example, use of a high salt elution buffer followed by dialysis into a lower salt buffer, or by changing pH (or other characteristics depending on the affinity matrix utilized), or competitive removal using a naturally occurring substrate of the affinity moiety.
• the methods are preferably non-injurious to the Pellino polypeptides.
• solutions containing Pellino polypeptides of the invention first can be incubated with a biotinylated Pellino binding partner. Incubation periods are typically at least one hour in duration to ensure sufficient binding to polypeptides of the invention.
• the resulting mixture then is passed through a column packed with avidin-coated beads, whereby the high affinity of biotin for avidin provides the binding of the Pellino polypeptides to the beads.
• avidin-coated beads is known in the art. See Berenson, et at. J. Cell. Biochem., 10D:239 (1986). Washing of unbound material and the release of the bound cells are performed using conventional methods.
• the desired degree of purity depends on the intended use of the protein.
• a relatively high degree of purity is desired when the polypeptide is to be administered in vivo, for example.
• the polypeptides are purified such that no protein bands corresponding to other proteins are detectable upon analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). It will be recognized by one skilled in the pertinent field that multiple bands corresponding to the polypeptide may be visualized by SDS-PAGE, due to differential glycosylation, differential post-translational processing, and the like.
• the polypeptide of the invention is purified to substantial homogeneity, as indicated by a single protein band upon analysis by SDS-PAGE.
• the protein band may be visualized by silver staining, Coomassie blue staining, or (if the protein is radiolabeled) by autoradiography.
• nucleic acids of the invention are useful as probes or primers.
• Such fragments generally comprise at least about 17 contiguous nucleotides of a DNA sequence.
• a DNA fragment comprises at least 30, or at least 60, contiguous nucleotides of a DNA sequence.
• probes based on the DNA sequence of SEQ ID NOs:1, 3, 5. 7, or 11 may be used to screen cDNA libraries derived from other mammalian species, using conventional cross-species hybridization techniques.
• oligonucleotides are useful as primers, e.g., in polymerase chain reactions (PCR), whereby DNA fragments are isolated and amplified.
• PCR polymerase chain reactions
• All or a portion of the nucleic acids of SEQ ID NOs:1, 3, 5, 7, or 11, including oligonucleotides can be used by those skilled in the art using well-known techniques to identify the human chromosome, and the specific locus thereof, that contains the DNA of a Pellino family member.
• Useful techniques include, but are not limited to, using the sequence or portions, including oligonucleotides, as a probe in various well-known techniques such as radiation hybrid mapping (high resolution), in situ hybridization to chromosome spreads (moderate resolution), and Southern blot hybridization to hybrid cell lines containing individual human chromosomes (low resolution).
• chromosomes can be mapped by radiation hybridization, using primers that lie within a putative exon of the gene of interest and which amplify a product from human genomic DNA, but do not amplify genomic DNA from other species.
• the results of the PCR are converted into a data vector that is scored and the chromosomal assignment and placement relative to known Sequence Tag Site (STS) markers on the radiation hybrid map is determined.
• STS Sequence Tag Site
• nucleic acid of SEQ ID NOs 1, 3, 5, 7, or 11, or a fragment thereof can be used by one skilled in the art using well-known techniques to analyze abnormalities associated with gene mapping to a chromosome that comprises a gene encoding Pellino. This enables one to distinguish conditions in which this marker is rearranged or deleted.
• nucleotides of SEQ ID NOs:1, 3, 5, 7, or 11, or a fragment thereof can be used as a positional marker to map other genes of unknown location.
• the DNA may be used in developing treatments for any disorder mediated (directly or indirectly) by defective, or insufficient amounts of, the genes corresponding to the nucleic acids of the invention. Disclosure herein of native nucleotide sequences permits the detection of defective genes, and the replacement or supplementation thereof with normal genes, by various gene therapy techniques that are known in the art. Defective genes may be detected in in vitro diagnostic assays, and by comparison of a native nucleotide sequence disclosed herein with that of a gene derived from a person suspected of harboring a defect in this gene.
• antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences.
• Antisense or sense oligonucleotides comprise a fragment of DNA of SEQ ID NOs:1, 3, 5, 7, or 11. Such a fragment generally comprises at least about 17 nucleotides, preferably from about 17 to about 30 nucleotides.
• Stein and Cohen Cancer Res.
• Binding of antisense or sense oligonucleotides to target nucleic acid sequences results in the formation of complexes that block or inhibit protein expression by one of several means, as discussed in U.S. Patent 5,783,665, issued July 21, 1998.
• Organic moieties and other moieties that increases affinity of the oligonucleotide for a target nucleic acid sequence, or intercalating agents, may be attached to sense or antisense oligonucleotides to modify binding specificities of the antisense or sense oligonucleotide for the target nucleotide sequence.
• the antisense or sense oligonucleotides may be introduced into a cell containing the target nucleic acid sequence by any gene transfer method, including, for example, lipofection, CaP04-mediated DNA transfection, electroporation, or by using gene transfer vectors such as Epstein-Barr virus.
• Sense or antisense oligonucleotides also may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753.
• a sense or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as described in WO 90/10448.
• inventive DNAs will also be useful in the development of transgenic and/or knockout cells and animals.
• Those of ordinary skill in the art are aware of various methods by which such cells or animals can be prepared; an exemplary method is given in U.S. Patent 5,565,321, issued October 15, 1996.
• the techniques described therein can be used with the inventive sequences by the application of routine experimentation.
• Pellino proteins are homologous to the Pellino proteins of Drosophila, an important molecule in the signaling cascade for the IL-IR/Toll family of receptors, small molecule inhibitors of its function or protein associations(or antisense or other inhibitors of its synthesis) may be useful in treating autoimmune and/or inflammatory disorders. Accordingly, the Pellino polypeptides of the present invention may be used in a screening assay to identify compounds and small molecules which inhibit (antagonize) or enhance (agonize) activation of the polypeptides of the instant invention.
• polypeptides of the invention may be used to identify antagonists and agonists from cells, cell-free preparations, chemical libraries, and natural product mixtures.
• the antagonists and agonists may be natural or modified substrates, ligands, enzymes, receptors, etc. of the polypeptides of the instant invention, or may be structural or functional mimetics of the polypeptides.
• Potential antagonists of the instant invention may include small molecules, peptides and antibodies that bind to and occupy a binding site of the inventive polypeptides or a binding partner thereof, causing them to be unavailable to bind to their natural binding partners and therefore preventing normal biological activity.
• Potential agonists include small molecules, peptides and antibodies which bind to the instant polypeptides or binding partners thereof, and elicit the same or enhanced biologic effects as those caused by the binding of the polypeptides of the instant invention.
• Small molecule agonists and antagonists are usually less than 10K molecular weight and may possess a number of physicochemical and pharmacological properties which enhance cell penetration, resist degradation and prolong their physiological half-lives (Gibbs, J., Pharmaceutical Research in Molecular Oncology. Cell, Vol. 79 (1994)).
• Antibodies which include intact molecules as well as fragments such as Fab and F(ab)2 fragments, as well as recombinant molecules derived therefrom, may be used to bind to and inhibit the polypeptides of the instant invention by blocking the propagation of a signaling cascade. It is preferable that the antibodies are humanized, and more preferable that the antibodies are human.
• the antibodies of the present invention may be prepared by any of a variety of well-known methods.
• Specific screening methods are known in the art and along with integrated robotic systems and collections of chemical compounds/natural products are extensively incorporated in high throughput screening so that large numbers of test compounds can be tested for antagonist or agonist activity within a short amount of time.
• These methods include homogeneous assay formats such as fluorescence resonance energy transfer, fluorescence polarization, time-resolved fluorescence resonance energy transfer, scintillation proximity assays, reporter gene assays, fluorescence quenched enzyme substrate, chromogenic enzyme substrate and electrochemiluminescence, as well as more traditional heterogeneous assay formats such as enzyme-linked immunosorbant assays (ELISA) or radioimmunoassays.
• ELISA enzyme-linked immunosorbant assays
• Homogeneous assays are "mix and read” assays that are very amenable to robotic application, whereas heterogeneous assays require separation of bound analyte from free by more complex unit operations such as filtration, centrifugation or washing. These assays are utilized to detect a wide variety of specific biomolecular interactions and the inhibition thereof by small organic molecules, including protein-protein, receptor-ligand, enzyme-substrate, etc. These assay methods and techniques are well known in the art and are described more fully in the following: High Throughput Screening: The Discovery of Bioactive Substances, John P.
• the screening assays of the present invention are amenable to high throughput screening of chemical libraries and are suitable for the identification of small molecule drug candidates, antibodies, peptides and other antagonists and/ or agonists.
• One embodiment of a method for identifying molecules which inhibit or antagonize the polypeptides involves adding a candidate molecule to a medium which contains cells that express the polypeptides of the instant invention; changing the conditions of said medium so that, but for the presence of the candidate molecule, the polypeptides would be bound to their natural ligands, substrates or effector molecules, and observing the binding and stimulation or inhibition of a functional response.
• the activity of the cells which were contacted with the candidate molecule may then be compared with the identical cells which were not contacted and antagonists and agonists of the polypeptides of the instant invention may be identified.
• the measurement of biological activity may be performed by a number of well-known methods such as measuring the amount of protein present (e.g. an ELISA) or of the proteins activity. A decrease in biological stimulation or activation would indicate an antagonist. An increase would indicate an agonist.
• Screening assays can further be designed to find molecules that mimic the biological activity of the polypeptides of the instant invention.
• Molecules which mimic the biological activity of a polypeptide may be useful for enhancing the biological activity of the peptide.
• To identify compounds for therapeutically active agents that mimic the biological activity of a polypeptide it must first be determined whether a candidate molecule binds to the polypeptide. A binding candidate molecule is then added to a biological assay to determine its biological effects. The biological effects of the candidate molecule are then compared to those of the polypeptide(s).
• Another embodiment of the invention relates to uses of Pellino polypeptides to study cell signal transduction.
• Cellular signaling often involves a molecular activation cascade, during which a receptor propagates a ligand-receptor mediated signal by specifically activating intracellular kinases which phosphorylate target substrates.
• These substrates can themselves be kinases which become activated following phosphorylation.
• they can be adaptor molecules that facilitate down stream signaling through protein-protein interaction following phosphorylation. Accordingly, these novel Pellino polypeptides can be used as reagents to identify novel molecules involved in signal transduction pathways.
• inventive polypeptides are involved in IL-1 signaling, and as such can be used as inhibitors of the IL-1 signaling pathway. Accordingly, they find utility in in vitro screening assays and in vivo therapeutics. As therapeutics that are cell membrane permeable, the Pellino polypeptides and fragments thereof can be administered to agonize or antagonize IL-IR mediated signaling pathways, thus providing useful immunoregulators.
• inventive polypeptides are envisioned herein.
• compositions of the present invention may contain a polypeptide in any form described herein, such as native proteins, variants, derivatives, oligomers, and biologically active fragments.
• the composition comprises a soluble polypeptide or an oligomer comprising soluble Pellino polypeptides.
• compositions comprising an effective amount of a polypeptide of the present invention, in combination with other components such as a physiologically acceptable diluent, carrier, or excipient, are provided herein.
• the polypeptides can be formulated according to known methods used to prepare pharmaceutically useful compositions. They can be combined in admixture, either as the sole active material or with other known active materials suitable for a given indication, with pharmaceutically acceptable diluents (e.g., saline, Tris-HCI, acetate, and phosphate buffered solutions), preservatives (e.g., thimerosal, benzyl alcohol, parabens), emulsifiers, solubilizers, adjuvants and/or catriers.
• Suitable formulations for pharmaceutical compositions include those described in Remington's Pharmaceutical Sciences, 16th ed. 1980, Mack Publishing Company, Easton, PA.
• compositions can be complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextran, etc., or incorporated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts.
• PEG polyethylene glycol
• metal ions or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextran, etc.
• liposomes such as polyacetic acid, polyglycolic acid, hydrogels, dextran, etc.
• Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance, and are thus chosen according to the intended application.
• compositions of the invention can be administered in any suitable manner, e.g., topically, parenterally, or by inhalation.
• parenteral includes injection, e.g., by subcutaneous, intravenous, or intramuscular routes, also including localized administration, e.g., at a site of disease or injury (for example, intracoronary or intra tumor administration or injection into a joint undergoing an inflammatory reaction). Sustained release from implants is also contemplated.
• suitable dosages will vary, depending upon such factors as the nature of the disorder to be treated, the patient's body weight, age, and general condition, and the route of administration. Preliminary doses can be determined according to animal tests, and the scaling of dosages for human administration is performed according to art-accepted practices.
• the polypeptide of the instant invention may also be administered by the method of protein transduction.
• the Pellino polypeptide is covalently linked to a protein-transduction domain (PTD) such as, but not limited to, TAT, Antp, or VP22 (Schwarze et al., 2000, Cell Biology 10: 290-295).
• PTD protein-transduction domain
• the PTD-linked Pellino polypeptides can then be transduced into cells by adding them to tissue-culture media containing the cells (Schwarze et al., 1999, Science 285: 1569; Lindgren et al., 2000, TiPS 21: 99; Derossi et al., 1998, Cell Biology 8: 84; WO 00/34308; WO 99/29721; and WO 99/10376).
• DNA encoding a polypeptide can be administered to a mammal in such a way that it is taken up by cells, and expressed. The resultant protein will then be available to exert a therapeutic effect.
• compositions comprising nucleic acids in physiologically acceptable formulations are also contemplated.
• DNA may be formulated for injection, for example.
• Antibodies that are immunoreactive with the polypeptides of the invention are provided herein. Such antibodies specifically bind to the polypeptides via the antigen-binding sites of the antibody (as opposed to non-specific binding).
• the polypeptides, fragments, variants, fusion proteins, etc., as set forth above may be employed as "immunogens" in producing antibodies immunoreactive therewith. More specifically, the polypeptides, fragment, variants, fusion proteins, etc. contain antigenic determinants or epitopes that elicit the formation of antibodies.
• Linear epitopes are composed of a single section of amino acids of the polypeptide, while conformational or discontinuous epitopes are composed of amino acids sections from different regions of the polypeptide chain that are brought into close proximity upon protein folding (C. A. Janeway, Jr. and P. Travers, Immuno Biology 3:9 (Garland Publishing Inc., 2nd ed. 1996)). Because folded proteins have complex surfaces, the number of epitopes available is quite numerous; however, due to the conformation of the protein and steric hindrances, the number of antibodies that actually bind to the epitopes is less than the number of available epitopes (C. A. Janeway, Jr. and P. Travers, Immuno Biology 2:14 (Garland Publishing Inc., 2nd ed. 1996)). Epitopes may be identified by any of the methods known in the art.
• one aspect of the present invention relates to the antigenic epitopes of the polypeptides of the invention.
• Such epitopes are useful for raising antibodies, in particular monoclonal antibodies, as described in more detail below.
• epitopes from the polypeptides of the invention can be used as research reagents, in assays, and to purify specific binding antibodies from substances such as polyclonal sera or supernatants from cultured hybridomas.
• Such epitopes or variants thereof can be produced using techniques well known in the art such as solid-phase synthesis, chemical or enzymatic cleavage of a polypeptide, or using recombinant DNA technology.
• both polyclonal and monoclonal antibodies may be prepared by conventional techniques. See, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980); and Antibodies: A Laboratory Manual. Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988).
• Hybridoma cell lines that produce monoclonal antibodies specific for the polypeptides of the invention are also contemplated herein. Such hybridomas may be produced and identified by conventional techniques.
• One method for producing such a hybridoma cell line comprises immunizing an animal with a polypeptide or a DNA encoding a polypeptide; harvesting spleen cells from the immunized animal; fusing said spleen cells to a myeloma cell line, thereby generating hybridoma cells; and identifying a hybridoma cell line that produces a monoclonal antibody that binds the polypeptide.
• the monoclonal antibodies may be recovered by conventional techniques.
• the monoclonal antibodies of the present invention include chimeric antibodies, e.g., humanized versions of murine monoclonal antibodies. Such humanized antibodies may be prepared by known techniques and offer the advantage of reduced immunogenicity when the antibodies are administered to humans.
• a humanized monoclonal antibody comprises the variable region of a murine antibody (or just the antigen binding site thereof) and a constant region derived from a human antibody.
• a humanized antibody fragment may comprise the antigen binding site of a murine monoclonal antibody and a variable region fragment (lacking the antigen-binding site) derived from a human antibody. Procedures for the production of chimeric and further engineered monoclonal antibodies include those described in Riechmann et al.
• Antigen-binding fragments of the antibodies which may be produced by conventional techniques, are also encompassed by the present invention.
• fragments include, but are not limited to, Fab and F(ab)2 fragments.
• Antibody fragments and derivatives produced by genetic engineering techniques are also provided.
• the antibodies are specific for the polypeptides of the present invention and do not cross-react with other proteins. Screening procedures by which such antibodies may be identified are well known, and may involve immunoaffinity chromatography, for example.
• the antibodies of the invention can be used in assays to detect the presence of the polypeptides or fragments of the invention, either in vitro or in vivo.
• the antibodies also may be employed in purifying polypeptides or fragments of the invention by immunoaffinity chromatography.
• the polynucleotide sequences of ESTs isolated from murine dendritic cells identified two clones containing open reading frames with a high degree of similarity to the Drosophila protein Pellino (Grosshans et al., supra).
• Appropriate flanking PCR primers were designed, and a novel nucleic acid was amplified from a murine cDNA library and cloned; the nucleotide sequence and encoded amino acid sequence of this clone, which is called Pellino-1 (previously referred to as conserveed Inflammatory Signal Target-1), are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.
• the amino acid sequence of the "human Pellino" polypeptide is identical to that of human Pellino-1 (SEQ ID NO:4) except for a Ser to Phe substitution at position 11.
• the difference between the human Pellino and SEQ ID NO:4 amino acid sequences may represent a naturally occurring allelic variation between nucleic acids encoding these amino acid sequences within the human population. Partial Pellino-1 amino acid sequences have also been published in WO 2000/58350; EP 1074 617; and WO 2001/09318.
• Pellino-2 a portion of a second, related gene, referred to as Pellino-2. was identified in the mouse and human.
• Pellino-2 amino acid sequences are 80% identical to their respective Pellino-l counterparts.
• Appropriate primers were designed, and murine Pellino-2 DNA was cloned substantially as described for Pellino-1; the nucleotide and amino acid sequence of murine Pellino-2 is shown in SEQ ID NO:5 and SEQ ID NO:6, respectively.
• the predicted nucleotide and amino acid sequence of human Pellino-2 is shown in SEQ ID NOs:7 and 8.
• a data set was received from Celera Genomics (Rockville, Maryland) containing a listing of amino acid sequences predicted to be encoded by the human genome. This data set was searched with a BLAST algorithm to identify Pellino polypeptide sequences and several partial amino acid sequences were found that appeared to be related to a new human Pellino polypeptide, Pellino-3. Comparison of these partial Pellino-3 amino acid sequences to genomic and cDNA sequences allowed the predicted human Pellino-3 nucleotide and amino acid sequences, SEQ ID NO:11 and SEQ ID NO:12, respectively, to be assembled. Two possible allelic variations have been detected within the human Pellino-3 amino acid sequence (SEQ ID NO: 12): a deletion of the Leu residue at position 96, and an Arg to Ala substitution at residue 353.
• Pellino- and -2 share 82% identity at the amino acid level, and the degree of conservation between human and mouse is extremely high; only one amino acid is different between human and mouse Pellino-l, and Pellino-2 is 95% conserved between these species.
• the predicted Pellino-3 amino acid sequence is 70% and 71% identical to human Pellino-1 and -2, respectively. There is a surprising degree of similarity between human Pellino-1, for example, and the homologous protein from C.
• Pellino-1 SEQ ID NOs 2 and 4; which corresponds to amino acids 134 through 195 in SEQ ID NO:8 and amino acids 158 through 219 in SEQ ID NO:12
• an absolutely conserved motif from residue 245 through residue 254 of SEQ ID NOs 2 and 4 (which corresponds to amino acids 247 through 256 in SEQ ID NO:8 and amino acids 271 through 280 in SEQ ID NO:12).
• the C-terminal portions of Pellino polypeptides are interspersed by a series of short, invariant motifs, in which cysteine, proline, histidine and large hydrophobic residues are prevalent.
• the Pellino RING-finger-like domains comprise the following amino acid sequences: amino acid 333 through amino acid 398 of SEQ ID NOs 2 and 4; amino acids 335 through 400 in SEQ ID NO:8 and the corresponding region of SEQ ID NO:6; and amino acids 360 through 425 in SEQ ID NO:12.
• the first cysteine following the conserved histidine of the canonical RING-finger domain is missing in Pellino, but we note that there is an almost invariant histidine at position 362 of SEQ ID NOs 2 and 4 (and at position 364 of SEQ ID NO:8 and the corresponding position in SEQ ID NO:6, and at positions 389 of SEQ ID NO:12) which might be available for co-ordination to a metal ion.
• a second, invariant Cys-Gly-His triplet at residues 311-313 of SEQ ID NOs 2 and 4 extends the zinc-finger resemblance further toward the N-terminus. Therefore, the C-terminal region of Pellino polypeptides appears to contain a novel type of Zinc finger-like domain.
• MsEPV Melanoplus sanguinipes Entomopoxvirus
• Amino acid substitutions and other alterations (deletions, insertions, etc.) to the Pellino amino acid sequences are predicted to be more likely to alter or disrupt Pellino polypeptide activities if they result in changes to the capitalized residues of the amino acid sequences as shown in Table 1, and particularly if those changes do not substitute a residue present in other Pellino polypeptides at that position in the alignment shown in Table 1.
• Embodiments of the invention include Pellino polypeptides and fragments of Pellino polypeptides comprising altered amino acid sequences.
• Altered Pellino polypeptide sequences share at least 30%, or more preferably at least 40%, or more preferably at least 50%, or more preferably at least 55%, or more preferably at least 60%, or more preferably at least 65%, or more preferably at least 70%, or more preferably at least 75%, or more preferably at least 80%, or more preferably at least 85%, or more preferably at least 90%, or more preferably at least 95%, or more preferably at least 97.5%, or more preferably at least 99%, or most preferably at least 99.5% amino acid identity with the Pellino amino acid sequences of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:12.
• the human Pellino-1, -2, and -3 coding sequences were compared with publicly available preliminary human genomic DNA sequences, and the following chromosome 2, 14, and 11 contigs were identified as containing human Pellino-1, -2, and -3 coding sequences, respectively: AC013466.3 (Pellino-1), AL138995.4 and AL355073.4 (Pellino-2), and AC027270.3 (Pellino-3).
• the approximate positions of the exons containing Pellino-1, -2, and -3 coding sequences in the above contigs are shown in the table below, along with their locations relative to SEQ ID NOs 3.
• the exons within the contig are not always in the right order or orientation with respect to each other, and may contain sequence variations due to inaccurate sequence data or allelic polymorphism.
• the genomic contig AC013466.3 has two copies of the Pellino-1 Exon 2 sequence present in opposite orientations with respect to each other, as indicated in the table below.
• the genomic sequences comprising human Pellino-1 exons map to the 2p13.3 region of human chromosome 2.
• Human Pellino nucleic acids such as SEQ ID NO:3 and fragments thereof are useful for the cytological identification of this chromosomal region, and for the genomic mapping of human genetic disorders such as the following disorders that have been mapped to this region: Preeclampsia/Eciampsia gene 1. Alstrom Syndrome. Parkinson Disease gene 3. Orofacial Cleft gene 2. and Welander Distal Myopathy.
• the genomic sequences comprising human Pellino-2 exons map to the 14q24.3 region of human chromosome 14.
• Human Pellino nucleic acids such as SEQ ID NO:7 and fragments thereof are useful for the cytological identification of this chromosomal region, and for the genomic mapping of human genetic disorders such as the following disorders that have been mapped to this region: Achromatopsia gene 1.
• the genomic sequences comprising human Pellino-3 exons map to a region of human chromosome 11 between 11p11.1 and 11q13, and are believed to map most closely to the 11q12.1 region.
• Human Pellino nucleic acids such as SEQ ID NO:3 and fragments thereof are useful for the cytological identification of this chromosomal region, and for the genomic mapping of human genetic disorders such as the following disorders that have been mapped to this region: Osteoporosis-Pseudoglioma Syndrome and Spinocerebellar Ataxia gene 5.
• the murine Pellino-1 coding sequence DNA was fused, in frame at the 3' end, to DNA encoding the FLAG epitope, followed by an in-frame stop codon.
• This construct was cloned into the mammalian expression vector pDC304 (identical to pDC302, described in U.S. Patent 5,599,905, issued February 4, 1997, except that the early splice region, consisting of splice donor and acceptor sites of the SV40 viral element, has been removed); and transfected into an IL-1-responsive line of COS-7 cells by the DEAE-dextran method.
• the cells were transiently transfected with Pellino test plasmid, 50 ng of reporter plasmid DNA, and empty vector, as required, to a total of 1 micrograms of total DNA per well. After 24 hours, stimulating agents were added in a small amount (less than 0.5% final volume) of medium or dimethyl sulfoxide, and the cells were re-incubated for 5 hours. Cells were lysed in luciferase Reporter Lysis Buffer (Promega, Madison WI., 0.25 ml per well) and luciferase activity was measured in a EG&G/Berthold luminometer after addition of Luciferase Assay Reagent (Promega) according to the supplier's instructions. All results were normalized to total protein content of the lysates as measured using the micro-BCA assay (Pierce, Rockford, IL).
• inhibitory effect of a preparation of Pellino-1-FLAG on NFkB-dependent reporter gene activity are the presence of mutated forms of the polypeptide as described below, or the relative presence or absence of a yet-to-be-characterized factor in a particular cell line.
• the wild-type Pellino-l-FLAG polypeptide had a stimulatory effect on IL-1-induced NF-kB-dependent reporter gene activity (see Table 2, below); wild-type Pellino-l-FT.AG also moderately augmented Jun N-terminal kinase, p38 kinase, and ERK signaling mediated by IL-1.
• wild-type Pellino-l polypeptide When expressed in COS-1 cells, wild-type Pellino-l polypeptide stimulates IL-8 promoter-reporter gene activity and NF-kB-dependent reporter activity in both the presence and absence of treatment with TNF-alpha, as compared to a vector-only control. In similar experiments, transfection of COS cells with moderate amounts of construct expressing wild-type Pellino-2 polypeptide also stimulates NF-kB-dependent reporter gene activity.
• Pellino-1 polypeptide was therefore chosen for mutation, since it might be expected that some of the resulting mutants would be resistant to cleavage, or otherwise altered in their response to pro-inflammatory stimuli.
• Cleavage of Pellino is sensitive to a chymotrypsin inhibitor, TPCK, and chymotrypsin has a requirement for a large, aromatic residue on one side of its cleavage site.
• TPCK chymotrypsin inhibitor
• chymotrypsin has a requirement for a large, aromatic residue on one side of its cleavage site.
• the Pellino-cleaving enzyme might share the same specificity determinants, we chose to mutate four of the aromatic amino acids that are invariably found in this region in the mammalian Pellino polypeptides.
• the two internal deletion mutants were chosen to flank the predicted cleavage site, and also to include highly conserved residues.
• a series of truncation mutants deleting residues from both amino- and carboxyl-termini, and mutants lacking conserved cysteine residues in the RING-finger-like domain, were constructed as follows.
• short sense-strand oligonucleotide primers were synthesized in which a sequence containing a KpnI restriction site and a methionine codon was fused to murine Pellino- sequences beginning with the codons for Gly-S1, Phe-100, Asp-181, and Val-231.
• PCR fragments which were subsequently cloned into pDC304 and referred to as encoding the 1-150, 1-250, and 1-350 mutants, respectively.
• the constructs encoding the single amino-acid substitutions F137L-FLAG, Y154A-FLAG, F158A-FLAG, and F165L-FLAG were constructed using the QuickChange site-directed mutagenesis kit (Stratagene, LaJolla, CA).
• d133-156-FLAG and d155-158-FLAG we made, in each case, a pair of PCR fragments containing a restriction site introduced to flank the sequence to be deleted.
• N-terminal deletion mutants of Pellino polypeptides having inhibitory activity on NF-kB-dependent transcription for example, a mutant in which N-terminal amino acids corresponding to the 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, or 175 N-terminal amino acids of SEQ ID NO:2 are deleted, but which still retains inhibitory activity.
• mutants within the N-terminal amino acids of Pellino polypeptides corresponding to the 180 N-terminal amino acids of SEQ ID NO:2 may be deleted in order to generate mutant forms of Pellino polypeptides having inhibitory activity on NF-kB-dependent transcription.
• mutants in which deletions (of one to 50 amino acids and more preferably one to 30 amino acids) are made within the central conserved domain and the RING-finger-like domain may also exhibit inhibitory activity on NF-kB-dependent transcription. All such mutants can be readily tested for activity using the reporter gene assays described herein. Table 2. Form of Pellino-1 Description Soluble or Insoluble Cleaved?
• Pellino polypeptides Because wild-type forms of Pellino polypeptides have stimulatory effects on key components of the four major MAP kinase-activated signaling pathways - stimulation of Jun N-terminal kinase, p38 kinase, and ERK signaling, and stimulation of NF-kB-dependent transcription - the "dominant-negative" mutant forms of Pellino polypeptides are expected to inhibit the Jun kinase and ERK MAP kinase-activated signaling pathways in a similar fashion to the inhibition of the p38 and NF- ⁇ B MAP kinase-activated signaling pathways.
• This example describes a method for monitoring the regulation by IL-1 (or other cytokine or molecule) of the intracellular localization of Pellino-1 in cells.
• COS-7 cells which express an endogenous IL-1 receptor
• COS-7 cells are transfected with an expression vector comprising FLAG-Pellino-1 as described in Example 2.
• Cells may also be transfected at the same time with other cDNAs encoding proteins which mediate inflammatory signaling, such as IL-1 Receptor-associated kinase (IRAK; GenBank NP001560).
• Transfected cells are cultured for about 48 hours.
• IL-1 (20ng/ml), or another cytokine or molecule at an appropriate concentration is added to the culture medium for the last 15 minutes (short-term stimulation) or 24 hours (prolonged stimulation) of this culture period.
• lysis buffer a buffer containing 50 mM Tris-chloride pH 8.0 supplemented with 1% nonidet (NP-40).
• sodium deoxycholate 0.5% sodium deoxycholate, 0.1 mM sodium orthovanadate, 30 mM para -nitrophenol phosphate, 30 mM beta-glycerophosphate, 140 mM NaCl, 5 mM dithiothreitol, 2mM EDTA, 10 mM leupeptin, 10 mM pepstatin A and 1 mM phenymethylsulfonyl fluoride; chemicals purchased from Sigma, St. Louis, MO). Solubilization of the cellular proteins can be facilitated by passage of the lysates several times through 25-gauge hypodermic needles. The lysate is centrifuged at 13,000xG at 4°C.
• the supernatant at this stage is referred to as the "soluble fraction.”
• the remaining pellet is solubilized in 1X SDS-PAGE sample buffer (Laemmli et al., Nature 227:680; 1970); this material is referred to as the "insoluble fraction.”
• COS7 monkey kidney cells were maintained in Dulbecco's modified Eagles medium containing 5% fetal bovine serum and supplemented with 100 units/ml penicillin and 100 micrograms/ml streptomycin at 35°C in 5% CO 2 .
• cells were scraped into 0.4 ml of an lysis/extraction buffer consisting of 50 mM Tris-HCl pH 7.8,1% NP-40, 0.15M NaCl. 2 mM EGTA, 5 microM NaF, 30 mM ⁇ -glycerophosphate, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 10 micrograms/ml leupeptin, and 10 micrograms/ml pepstatin A.
• an lysis/extraction buffer consisting of 50 mM Tris-HCl pH 7.8,1% NP-40, 0.15M NaCl. 2 mM EGTA, 5 microM NaF, 30 mM ⁇ -glycerophosphate, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 10 micrograms
• the cell suspensions were allowed to lyse on ice for 15 minutes and were centrifuged (13000 rpm for 10 minutes at 4 degrees C) in a microcentrifuge. The supernatants were carefully removed to fresh tubes and diluted with one-third volume of 4x-concentrated SDS-PAGE sample buffer containing 2-mercaptoethanol. The supernatant samples ('detergent-soluble' fraction) were boiled for 5 minutes. The pellets were re-extracted by resuspending them in one half the original volume of 1x-concentrated SDS-PAGE sample buffer, vortexing, and then boiling them.
• Matched aliquots of soluble fraction and insoluble fraction may then be analyzed by gel electrophoresis on 4-20% gradient SDS-polyacrylamide gels (Novex, Invitrogen Corp.,. Carlsbad CA), and transferred to nitrocellulose membranes, and assayed by western immunoblotting using anti-FLAG antibodies (i.e., FLAG M2) to bind to the protein products of the transfected cDNAs. Proteins are visualized by incubation of western blots with horseradish-peroxidase-conjugated anti-mouse IgG (BioRad, San Diego, CA) followed by detection using the ECL system (Amersham; Arlington Heights, IL).
• Transfected cells are prepared substantially as described above.
• the cells are co-transfected with a pDC304 vector containing a cDNA insert coding for human IRAK with a tandem 3' FLAG and poly-His 'tail.
• the cells are co-transfected with a catalytically inactive human FLAG-polyHis-IRAK expression vector in which lysine residue 293 in the ATP-binding pocket of IRAK is replaced with an alanine.
• Pellino-1-transfected cells were stimulated with agents that activate NF-kB by IRAK-independent pathways such as phorbol myristate acetate (PMA, 100 ng/ml), which is known to promote many of the same intracellular signals as IL-1, and TNF-alpha (20 ng/ml).
• PMA phorbol myristate acetate
• TNF-alpha activates NF-kB through a mechanism involving the adaptor protein TRADD, the kinase RIP, and TRAF2, while PMA stimulates the activity of protein kinase C which is known to cross-talk with the NF-kB pathway.
• EGF epidermal growth factor
• COS7 cells transfected with a Pellino-1-expressing construct were treated with PMA together with inhibitors of different classes of proteinases as shown in Table 3 below.
• Pellino-1 For a 30 kDa Pellino-1 fragment to be generated, proteolytic cleavage would be predicted to occur within the region of amino acids 132 to 189 of SEQ ID NO:2. Examination of the amino acid sequence of Pellino-1 shows it has been extremely well conserved in those species for which EST sequence data is available (murine and human Pellino-1 and Pellino-2, disclosed herein; Drosophila pellino, GenBank accession number AF091624; and the F25B4.2 gene product of Caenorhabditis elegans, GenBank accession number U64842). A number of conserved phenylalanine and tyrosine residues are present in this region, any of which might serve as the recognition site for a chymotrypsin-like serine protease.
• This example describes the construction and expression of a recombinant Glutathione S-transferase (GST)-Pellino-1 fusion protein.
• PCR primers were synthesized having the sequences ATATTCACTGAATTCTGATGTTTTCTCCTGATCAA (Primer 1; SEQ ID NO:9) and AGTGAATATGAATTCCTACTTATCATCGTCATCTTTG (Primer 2; SEQ ID NO:10), for the sense and antisense primers, respectively. These were designed for use with a Pellino-1-FLAG template, and add a EcoR1 site to each end of the amplified product.
• This PCR product was ligated into the unique EcoR1 cloning site of the vector pGEX2T (described in EP 0293249-A) such that the coding sequences of the glutathione S-transferase gene and FLAG-Pellino-1 were in the same frame.
• E coli strain DH10B were transformed with the resultant vector and a one-liter culture was grown. Transcription of the GST-Pellino-1-FLAG gene was induced by addition of IPTG (0.1 mM) to the bacterial culture for three hours.
• Bacterial cells were harvested and lysed according to methods well known in the art (see, for example, Smith D.B., Johnson K.S.; Gene 67:31-40(1988)). The lysate containing solubilized GST-Pellino-l-FLAG was purified on 1 ml of Glutathione-Agarose beads (Pharmacia), according to the directions supplied by the manufacturer.
• This example illustrates the preparation of monoclonal antibodies against Pellino polypeptides.
• Preparations of purified recombinant Pellino polypeptides, for example, or transfected cells expressing high levels of Pellino polypeptides are employed to generate monoclonal antibodies against Pellino polypeptides using conventional techniques, such as those disclosed in U.S. Patent 4,411,993.
• DNA encoding Pellino polypeptides can also be used as an immunogen, for example, as reviewed by Pardoll and Beckerleg in Immunity 3:165, 1995.
• Such antibodies are likely to be useful as components of diagnostic or research assays for Pellino or Pellino activity, or in affinity purification of Pellino polypeptides.
• Pellino immunogen for example, a Pellino-1 peptide comprising amino acids 2 through 20, amino acids 118 through 131, or amino acids 318 through 340 of SEQ ID NOs 2 and 4
• an immunogenic molecule such as keyhole limpet hemocyanin
• an adjuvant such as complete or incomplete Freund's adjuvant, alum, or another adjuvant, such as Ribi adjuvant R700 (Ribi, Hamilton, MT)
• Ribi adjuvant R700 Ribi, Hamilton, MT
• DNA may be given intradermally (Raz et al., Proc. Natl.
• saline has been found to be a suitable diluent for DNA-based antigens.
• the immunized animals are boosted with additional immunogen and periodically boosted thereafter on a weekly, biweekly or every third week immunization schedule.
• Serum samples are periodically taken by retro-orbital bleeding or tail-tip excision for testing by dot-blot assay (antibody sandwich), ELISA (enzyme-linked immunosorbent assay), immunoprecipitation, or other suitable assays, including FACS analysis.
• dot-blot assay antibody sandwich
• ELISA enzyme-linked immunosorbent assay
• immunoprecipitation or other suitable assays, including FACS analysis.
• positive animals are given an intravenous injection of antigen in saline. Three to four days later, the animals are sacrificed, splenocytes harvested, and fused to a murine myeloma cell line (e.g., NS 1 or preferably Ag 8.653 [ATCC CRL 1580]).
• a murine myeloma cell line e.g., NS 1 or preferably Ag 8.653 [ATCC CRL 1580]
• Hybridoma cell lines generated by this procedure are plated in multiple microtiter plates in a selective medium (for example, one containing hypoxanthine, aminopterin, and thymidine, or HAT) to inhibit proliferation of non-fused cells, myeloma-myeloma hybrids, and splenocyte-splenocyte hybrids.
• a selective medium for example, one containing hypoxanthine, aminopterin, and thymidine, or HAT
• Hybridoma clones thus generated can be screened by ELISA for reactivity with Pellino polypeptides, for example, by adaptations of the techniques disclosed by Engvall et al., Immunochem. 8:871 (1971) and in U.S. Patent 4,703,004.
• a preferred screening technique is the antibody capture technique described by Beckman et al., J. Immunol. 144:4212 (1990). Positive clones are then injected into the peritoneal cavities of syngeneic rodents to produce ascites containing high concentrations (>1 mg/ml) of anti-Pellino monoclonal antibody.
• the resulting monoclonal antibody can be purified by ammonium sulfate precipitation followed by gel exclusion chromatography.
• affinity chromatography based upon binding of antibody to protein A or protein G can also be used, as can affinity chromatography based upon binding to Pellino polypeptide.
• a 234-bp PCR fragment corresponding to the predicted 3'end of the human Pellino-1 mRNA was generated by standard amplification methods.
• the PCR fragment was purified (Qiagen PCR purification kit) and labeled by the Gibco Random Prime Oligonucleotide DNA Labeling kit.
• the cDNA riboprobe was denatured at 100 degrees C for 5 minutes and placed on ice.
• the cDNA probe was denatured at 100 degrees C for 5 minutes before being added to the hybridization solution.
• a multi-tissue northern blot containing RNA from human tissues - brain, heart, skeletal muscle, colon (no mucosa), thymus, spleen, kidney, liver, small intestine, placenta, lung, and peripheral blood leukocytes - was purchased from Clonetech (Palo Alto, CA).
• the blot was blocked in 5mL ExpressHyb Solution for 30 minutes at 68 degrees C.
• Fresh ExpressHyb solution containing the denatured radiolabeled cDNA probe was added to the membrane and incubated at 68 degrees C for I hour with continuous shaking.
• the blot was rinsed in 2X SSC, 0.05% SDS with four changes at room temperature for 40 minutes, followed by a wash in 0.1X SSC.
• the riboprobe for Pellino-1 hybridized to the Northern blot with a major band at 4.4 kilobases in all tissues represented, with an increased level of expression evident in peripheral blood leukocytes, lung, placenta, liver, kidney, skeletal muscle, and brain. The message appears to be more highly expressed in peripheral blood leukocytes, and in this tissue there seems to be two additional bands at 7.5 and 9.5 kb which do not appear in the lanes for the other tissues.
• the cDNA for human Pellino-1 is predicted to include 1251 base pairs in the coding region and 1931 base pairs in the 3' untranslated region, and no evidence for alternative splicing has yet been identified.
• SEQ ID NO:1 Nucleotide Murine (Mus musculus) Pellino- coding sequence SEQ ID NO:2 Amino acid Murine (Mus musculus) Pellino- amino acid sequence SEQ ID NO:3 Nucleotide Human (Homo sapiens) Pellino- coding sequence SEQ ID NO:4 Amino acid Human (Homo sapiens) Pellino-1 amino acid sequence SEQ ID NO:5 Nucleotide Murine (Mus musculus) Pellino-2 coding sequence SEQ ID N0:6 Amino acid Murine (Mus musculus) Pellino-2 amino acid sequence SEQ ID NO:7 Nucleotide Human (Homo sapiens) Pellino-2 coding sequence SEQ ID NO: 8 Amino acid Human (Homo sapiens) Pellino-2 amino acid sequence SEQ ID NO:9 Nucleotide PCR primer SEQ ID NO:10 Nucleotide PCR primer SEQ ID NO:

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